Oil in water emulsions containing saponins

ABSTRACT

A composition comprising an oil in water emulsion and a saponin, wherein said oil is a metabolisable oil, and having an advantageous ratio of metabolisable oil:saponin (w/w).

The present invention relates to an oil in water emulsion vaccinecomposition. In particular, the present invention relates to a vaccineadjuvant formulation based on oil in water emulsion comprising ametabolisable oil and a saponin. The invention further relates tomethods for preparing the emulsion and its use in medicine.

Induction of cytotoxic T-cell (CTL) responses occurs naturally duringinfection of a target cell, or uncontrolled synthesis of a tumourantigen, wherein enzymatic degradation of the target antigen takes placein the cell cytoplasm. This phenomenon allows cytoplasmic peptidesderived from the pathogen, or tumour specific antigen, to enter the Th1(endogenous antigen processing) pathway and be presented on the surfaceof the cell associated with an MHC class 1 molecule. If a vaccineantigen does not enter into the cytoplasm of the host cell, then itmight be taken up by the cell and enter the exogenous antigen processingpathway and ultimately be presented on the surface of the cellassociated with a MHC class II molecule. This alternative routegenerally results in T-helper responses and antigen specific antibodyresponses.

After conventional vaccination with subunit or non-living vaccines,antigen generally does not enter the cytoplasm of a host cell, andtherefore will not enter the endogenous antigen processing pathway andultimately will not induce a CTL response. CTL induction is believed tocorrelate with Th-1 cytokine profile responses, specifically with IFN-γand IL-2 secretion. IFN-γ secretion is associated with protectiveresponses against intracellular pathogens, including parasites, bacteriaand viruses. Activation of leucocytes by IFN-γ enhances killing ofintracellular pathogens and increases expression of Fc receptors. Directcytotoxicity may also occur, especially in synergy with lymphotoxin(another product of TH1 cells). IFN-γ is also both an inducer and aproduct of NK cells, which are major innate effectors of protection. TH1type responses, either through IFN-γ or other mechanisms, providepreferential help for murine IgG2a and human IgG1 immunoglobulinisotypes.

International patent application No. WO 95/17210 discloses an adjuvantemulsion system based on squalene, α-tocopherol, and polyoxyethylenesorbitan monooleate (TWEEN80), formulated with the immunostimulant QS21,optionally with 3D-MPL. This adjuvant formulation is a very potentinducer of a wide range of immune responses.

Immunologically active saponin fractions (e.g Quil A) having adjuvantactivity derived from the bark of the South American tree QuillajaSaponaria Molina are known in the art. Derivatives of Quil A, forexample QS21 (an HPLC purified fraction derivative of Quil A), and themethod of its production is disclosed in U.S. Pat. No. 5,057,540.Amongst QS21 (known as QA21) other fractions such as QA17 are alsodisclosed. The use of such saponins in isolation is accompanied withdisadvantage in that local necrosis, that is to say, localised tissuedeath, occurs at the injection site, thereby leading to pain.

Immunologically active saponin fractions having adjuvant activityderived from the bark of the South American tree Quillaja SaponariaMolina are known in the art. For example, QS21, an HPLC purifiedfraction from the Quillaja Saponaria Molina tree, and the method of itsproduction (known as QA21) is disclosed in U.S. Pat. No. 5,057,540. Theuse of such saponins is accompanied with a disadvantage in that localnecrosis, that is to say, localised tissue death, occurs at theinjection site, which leads to pain.

3 De-O-acylated monophosphoryl lipid A is a well known adjuvantmanufactured by Ribi Immunochem, Montana. Chemically it is oftensupplied as a mixture of 3 De-O-acylated monophosphoryl lipid A witheither 4, 5, or 6 acylated chains. It can be prepared by the methodstaught in GB 2122204B. A preferred form of 3 De-O-acylatedmonophosphoryl lipid A is in the form of an emulsion having a smallparticle size less than 0.2 μm in diameter, and its method ofmanufacture is disclosed in European Patent No. EP 0 671 948 B1.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system has to comprise ametabolisable oil. The meaning of the term metabolisable oil is wellknown in the art. Metabolisable can be defined as “being capable ofbeing transformed by metabolism” (Dorlarid's Illustrated MedicalDictionary, W.B. Sanders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts, seeds, and grains are common sources of vegetableoils. Synthetic oils are also part of this invention and can includecommercially available oils such as NEOBEE® and others. Squalene(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is anunsaturated oil which is found in large quantities in shark-liver oil,and in lower quantities in olive oil, wheat germ oil, rice bran oil, andyeast, and is a particularly preferred oil for use in this invention.Squalene is a metabolisable oil virtue of the fact that it is anintermediate in the biosynthesis of cholesterol (Merck index, 10thEdition, entry no. 8619).

Oil in water emulsions per se are well known in the art, and have beensuggested to be useful as adjuvant compositions (EPO 399843).

The immunological responses to administration of antigen formulated inthe oil in water emulsions described in International patent applicationNo. WO 95/17210, can be characterised in that both Th2 and Th1 responsesare observed. Given that for in many cases Th1-type responses have beenidentified as critical for the prophylaxis and treatment of disease, itis, therefore, desirable that a more Th1 biased adjuvant is developed.This has most surprisingly been achieved by the present inventors not byaddition of extra immunostimulators, but by a reduction of one of theoriginal components.

The oil in water adjuvant emulsions described in International patentapplication No. WO 95/17210 have a high ratio of squalene:saponin (w/w)of 240:1. The embodiments of the present invention have the ratio ofsqualene:QS21 in the range of 1:1 to 200:1, also preferred is the range20:1 to 200:1, preferably 20:1 to 100:1, and most preferablysubstantially 48:1. This reduction of one of the components has thesurprising effect of qualitatively improving the resultant immuneresponse. Using this novel adjuvant formulation strong Th2-typeresponses are maintained, but moreover such formulations elicit anenhanced immune response specifically associated with Th1-typeresponses, characterised by high IFN-γ, T-cell proliferative and CTLresponses.

One preferred embodiment of the present invention is an adjuvant orpharmaceutical formulation comprising QuilA or derivative thereof, suchas QS21 and an oil in water emulsion, wherein the oil in water emulsioncomprises a metabolisible oil, such as squalene, and a polysorbate(including polyoxyethylene sorbitan monooleate, TWEEN 80™), saidemulsions being characterised in that the ratio of the oil:QS21 is inthe range of 20:1 to 200:1 (w/w). In another preferred embodiment, theadjuvant formulation further comprises other immunomodulators, includingα-tocopherol and 3D-MPL.

Such formulations once combined with an antigen or antigenic preparationis suitable for a broad range of monovalent or polyvalent vaccines.Additionally the oil in water emulsion may contain polyoxyethylenesorbitan trioleate (SPAN 85). A preferred form of 3 De-O-acylatedmonophosphoryl lipid A is disclosed in International patent applicationpublished under No. 92116556—SmithKline Beecham Biologicals s.a.

Preferably the vaccine formulations of the present invention contain anantigen or antigenic composition capable of eliciting an immune responseagainst a human pathogen, which antigen or antigenic composition isderived from HIV-1, (such as tat, nef, gp120 or gp160), human herpesviruses, such as gD or derivatives thereof or Immediate Early proteinsuch as ICP27 from HSV1 or HSV2, cytomegalovirus ((esp Human) (such asgB or derivatives thereof), Rotavirus (including live-attenuatedviruses), Epstein Barr virus (such as gp350 or derivatives thereof),Varicella Zoster Virus (such as gpI, II and IE63), or from a hepatitisvirus such as hepatitis B virus (for example Hepatitis B Surface antigenor a derivative thereof), hepatitis A virus, hepatitis C virus andhepatitis E virus, or from other viral pathogens, such asparamyxoviruses: Respiratory Syncytial virus (such as F and G proteinsor derivatives thereof), parainfluenza virus, measles virus, mumpsvirus, human papilloma viruses (for example HPV6, 11, 16, 18, . . . ),flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borneencephalitis virus, Japanese Encephalitis Virus) or Influenza virus, orderived from bacterial pathogens such as Neisseria spp, including N.gonorrhea and N. meningitidis (for example capsular polysaccharides andconjugates thereof, transferrin-binding proteins, lactoferrin bindingproteins, PilC, adhesins); Streptococcus spp, including S. pneumoniae(for example capsular polysaccharides and conjugates thereof, PsaA,PspA, streptolysin, choline-binding proteins), S. pyogenes (for exampleM proteins or fragments thereof, C5A protease, lipoteichoic acids), S.agalactiae, S. mutans; Haemophilus spp, including H. influenzae type B(for example PRP and conjugates thereof), non typeable H. influenzae(for example OMP26, high molecular weight adhesins, P5, P6, lipoproteinD), H. ducreyi; Moraxella spp, including M catarrhalis, also known asBranhamella catarrhalis (for example high and low molecular weightadhesins and invasins); Bordetella spp, including B. pertussis (forexample pertactin, pertussis toxin or derivatives thereof, filamenteoushemagglutinin, adenylate cyclase, fimbriae), B. parapertussis and B.bronchiseptica; Mycobacterium spp., including M. tuberculosis (forexample ESAT6, Antigen 85A, -B or -C), M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof,heat-stable toxin or derivatives thereof), enterohemorragic E. coli,enteropathogenic E. coli (for example shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof), Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and C. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or derivedfrom parasites such as Plasmodium spp., including P. falciparum;Toxoplasma spp., including T. gondii (for example SAG2, SAG3, Tg34);Entamoeba spp., including E. histolytica; Babesia spp., including B.microti; Trypanosoma spp., including T. cruzi; Giardia spp., includingG. lamblia; Leshmania spp., including L. major; Pneumocystis spp.,including P. carinii; Trichomonas spp., including T. vaginalis;Schisostoma spp., including S. mansoni, or derived from yeast such asCandida spp., including C. albicans; Cryptococcus spp., including C.neoformans.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, PreS2 S antigens set forth describedin European Patent applications EP-A-414 374; EP-A-0304 578, and EP198-474. In one preferred aspect the vaccine formulation of theinvention comprises the HIV-1 antigen, gp120, especially when expressedin CHO cells. In a further embodiment, the vaccine formulation of theinvention comprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention vaccines containingthe claimed adjuvant comprise the HPV viruses considered to beresponsible for genital warts, (HPV 6 or HPV 11 and others), and the HPVviruses responsible for cervical cancer (HPV16, HPV18 and others).Particularly preferred forms of vaccine comprise L1 particles orcapsomers, and fusion proteins comprising one or more antigens selectedfrom the HPV 6 and HPV 11 proteins E6, E7, L1, and L2. The mostpreferred forms of fusion protein are: L2E7 as disclosed in GB 9515478.7, and proteinD(1/3)-E7 disclosed in GB 9717953.5.

Vaccines of the present invention further comprise antigens derived fromparasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. It's full structure is disclosed inthe International Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent application No.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S. TRAP antigens are described inthe International Patent Application No. PCT/GB89/00895, published underWO 90/01496. A preferred embodiment of the present invention is aMalaria vaccine wherein the antigenic preparation comprises acombination of the RTS,S and TRAP antigens. Other plasmodia antigensthat are likely candidates to be components of a multistage Malariavaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1, RAP2,Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25,Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp.

The formulations may also contain an anti-tumour antigen and be usefulfor the immunotherapeutic treatment cancers. For example, the adjuvantformulation finds utility with tumour rejection antigens such as thosefor prostrate, breast, colorectal, lung, pancreatic, renal or melanomacancers. Exemplary antigens include MAGE 1 and MAGE 3 or other MAGEantigens for the treatment of melanoma, PRAME, BAGE or GAGE (Robbins andKawakami, 1996, Current Opinions in Immunology 8, pps 628-636; Van denEynde et al., International Journal of Clinical & Laboratory Research(submitted 1997); Correale et al. (1997), Journal of the National CancerInstitute 89, p 293. Indeed these antigens are expressed in a wide rangeof tumour types such as melanoma, lung carcinoma, sarcoma and bladdercarcinoma. Other Tumor-Specific antigens are suitable for use withadjuvant of the present invention and include, but are not restricted toProstate specific antigen (PSA) or Her-2/neu, KSA (GA733), MUC-1 andcarcinoembryonic antigen (CEA). Accordingly in one aspect of the presentinvention there is provided a vaccine comprising an adjuvant compositionaccording to the invention and a tumour rejection antigen.

It is foreseen that compositions of the present invention will be usedto formulate vaccines containing antigens derived from Borrelia sp. Forexample, antigens may include nucleic acid, pathogen derived antigen orantigenic preparations, recombinantly produced protein or peptides, andchimeric fusion proteins. In particular the antigen is OspA. The OspAmay be a full mature protein in a lipidated form virtue of the host cell(E. Coli) termed (Lipo-OspA) or a non-lipidated derivative. Suchnon-lipidated derivatives include the non-lipidated NS1-OspA fusionprotein which has the first 81 N-terminal amino acids of thenon-structural protein (NS1) of the influenza virus, and the completeOspA protein, and another, MDP-OspA is a non-lipidated form of OspAcarrying 3 additional N-terminal amino acids.

Vaccines of the present invention may be used for the prophylaxis ortherapy of allergy. Such vaccines would comprise allergen specific (forexample Der p1) and allergen non-specific antigens (for example thestanworth decapeptide).

The ratio of QS21:3D-MPL (w/w) in a preferred embodiment of the presentinvention will typically be in the order of 1:10 to 10:1; preferably 1:5to 5:1 and often substantially 1:1. The preferred range for optimalsynergy is from 2.5:1 to 1:1 3D MPL: QS21. Typically, the dosages ofQS21 and 3D-MPL in a vaccine for human administration will be in therange 1 μg-1000 μg, preferably 10 μg-500 μg, and most preferably 10-100μg per dose. Typically the oil in water will comprise from 2 to 10%squalene, from 2 to 10% α-tocopherol and from 0.4 to 2% polyoxyethylenesorbitan monooleate (TWEEN 80). Preferably the ratio of squalene:α-tocopherol is equal or less than 1 as this provides a more stableemulsion. Polyoxyethylene sorbitan trioleate (SPAN 85) may also bepresent at a level of 0.5-1%. In some cases it may be advantageous thatthe vaccines of the present invention will further contain a stabiliser,for example other emulsifiers/surfactants, including caprylic acid(merck index 10th Edition, entry no. 1739), of which Tricaprylin is aparticularly preferred embodiment.

Therefore, another embodiment of this invention is a vaccine containingQS21 and an oil in water emulsion falling within the desired ratio,which is formulated in the presence of a sterol, preferably cholesterol,in order to reduce the local reactogenicity conferred by the QS21. Theratio of the QS21 to cholesterol (w/w), present in a specific embodimentof the present invention, is envisaged to be in the range of 1:1 to1:20, substantially 1:10.

The previous emulsions used in International patent application No. WO95/17210 obviously holds some advantages over conventional non-Th1inducing adjuvants. However, the inclusion of QS21 has so far made thispotent adjuvant reactogenic, thereby, leading to pain. It has beenobserved that formulation of the QS21 into cholesterol containingliposomes may help prevent the necrosis occurring at the site ofinjection. This observation is subject to the International PatentApplication No. PCT/EP96/01464.

In embodiments of the present invention the preferred sterol ischolesterol. Other sterols which could be used in embodiments of thepresent invention include β-sitosterol, stigmasterol, ergosterol,ergocalciferol and cholesterol. Sterols are well known in the art.Cholesterol is well known and is, for example, disclosed in the MerckIndex, 11th Edn., page 341, as a naturally occurring sterol found inanimal fat.

QS21 in aqueous solution is known to degenerate over time into anadjuvant-inactive form, QS21-H, which degeneration is mediated by ⁻OHhydrolysis by the aqueous medium. Such degeneration may occur when theQS21 is present in the aqueous phase of an oil in water adjuvant.Surprisingly it has been found that the addition of cholesterol to theoil phase of the oil in water emulsion has the effect of maintaining theQS21 in its active form, with obvious benefits to the shelf-life of theadjuvant/vaccine formulation. The present invention provides a method ofstablilising a preparation of a saponin, preferably QS21, in itsnon-hydrolysed adjuvant-active form, when the QS21 is present in an oilin water emulsion based adjuvant. This method comprises the addition ofa sterol, preferably cholesterol, into the oil phase of an oil in wateremulsion.

Such preparations are used as vaccine adjuvant systems and onceformulated together with antigen or antigenic preparations for potentvaccines. Advantageously they preferentially induce a Th1 response.

The emulsion systems of the present invention have a small oil dropletsize in the sub-micron range. Preferably the oil droplet sizes will bein the range 120 to 750 nm, and most preferably from 120-600 nm indiameter.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise 1-1000 μg of protein,preferably 1-500 μg, preferably 1-100 μg, most preferably 1 to 50 μg. Anoptimal amount for a particular vaccine can be ascertained by standardstudies involving observation of appropriate immune responses insubjects. Following an initial vaccination, subjects may receive one orseveral booster immunisation adequately spaced.

The formulations of the present invention maybe used for bothprophylactic and therapeutic purposes. In a further aspect of thepresent invention there is provided a vaccine as herein described foruse in medicine. Vaccine preparation is generally described in NewTrends and Developments in Vaccines, edited by Voller et al., UniversityPark Press, Baltimore, Md., U.S.A. 1978.

It is foreseen that compositions of the present invention will be usedto formulate vaccines containing antigens derived from a wide variety ofsources. For example, antigens may include human, bacterial, or viralnucleic acid, pathogen derived antigen or antigenic preparations, tumourderived antigen or antigenic preparations, host-derived antigens,including the histamine releasing decapeptide of IgE (known as theStanworth decapeptide), recombinantly produced protein or peptides, andchimeric fusion proteins.

The vaccine preparations of the present invention may be used to protector treat a mammal susceptible to, or suffering from a disease, by meansof administering said vaccine via systemic or mucosal route. Theseadministrations may include injection via the intramuscular,intraperitoneal, intradermal or subcutaneous routes; or via mucosaladministration to the oral/alimentary, or respiratory tracts.

EXAMPLE 1 Preparation of the Oil in Water Emulsion Adjuvants

The oil in water emulsion adjuvant formulations used in the subsequentexamples were each made comprising the following oil in water emulsioncomponent: 5% Squalene, 5% α-tocopherol, 2.0% polyoxyethylene sorbitanmonooleate (TWEEN 80).

The emulsion was prepared as follows as a 2 fold concentrate. Allexamples used in the immunological experiments are diluted with theaddition of extra components and diluents to give either a 1×concentration (equating to a squalene:QS21 ratio (w/w) of 240:1) orfurther dilutions thereof.

Briefly, the TWEEN 80 is dissolved in phosphate buffered saline (PBS) togive a 2% solution in the PBS. To provide 100 ml of a two foldconcentrate emulsion, 5 ml of DL alpha tocopherol and 5 ml of squaleneare vortexed to mix thoroughly. 95 ml of PBS/TWEEN solution is added tothe oil and mixed thoroughly. The resulting emulsion is then passedthrough a syringe needle and finally microfluidised by using an M110SMicrofluidics machine. The resulting oil droplets have a size ofapproximately 145-180 nm (expressed as z av. measured by PCS) and istermed “full dose” SB62.

The other adjuvant/vaccine components (QS21, 3D-MPL or antigen) areadded to the emulsion in simple admixture.

The antigen containing vaccines used herein are formulated either withfull dose SB62 adjuvant to give a high squalene:QS21 ratio (240:1) orwith a lower amount of SB62 to give a low ratio formulation (48:1),these adjuvant formulations are called SB62 and SB62′ respectively.Other vaccines may optionally be formulated with the addition ofcholesterol to the oil phase of the emulsion (denoted by the addition ofthe letter “c”).

EXAMPLE 2 Immunogenicity Studies with Recombinant Antigen S,L*

A study was conducted in Balb/C mice in order to compare theimmunogenicity of various S,L* containing formulations. S,L* is acomposite antigen comprising a modified surface antigen L protein (L*)and an S-protein both derived from the Hepatitis B virus (HB. Thiscomposite antigen is the subject of European Patent application No. EP 0414 374. This immunisation scheme used in the HBs transgenic mouse modelwhich has been shown previously to support the induction of CTL inBalb/c mice.

Different adjuvant formulations, using the emulsion systems as describedin example 1, with differing ratios of squalene:QS21, and optionallycomprising cholesterol (QS21:cholesterol ratio w/w of 1:10), werecombined with S,L* and compared in their ability to induce humoral andcell mediated immune responses (cytokine production and CTL). S,L*adsorbed onto Aluminium hydroxide (AlOH₃) was used as a Th2 inducingcontrol.

Briefly, groups of 10 mice were immunised intramuscularly 4 times at 3weeks interval with 2 μg lyophilised S,L* combined with various oil inwater emulsion systems (SB62). 14 days following the fourth immunisationthe production of cytokines (IL5 and IFN-γ) and CTL activity wasanalysed after in vitro restimulation of spleen and lymph nodes cellswith S,L* antigen. Antibody response to S,L* and the isotypic profileinduced were monitored by ELISA at 21 days post II and 14 days post IV.

Groups of Mice

Groups of 10 Balb/C mice were immunised intramuscularly withformulations described below. SB62 was formulated together with theantigen at a high (240:1, SB62) or low (48:1, SB62′) ratio ofsqualene:QS21, optionally with the addition of cholesterol (c).

TABLE 1 Groups of mice and vaccines compositions used in example 2.Antigen Adjuvant Group S,L* name Composition of adjuvant formulation GR1 2 μg SB62 25 μl SB62/5 μg QS21/5 μg 3D-MPL GR 2 2 μg SB62c 25 μlSB62c/5 μg QS21/5 μg 3D-MPL GR 3 2 μg SB62′ 5 μl SB62/5 μg QS21/5 μg3D-MPL GR 4 2 μg SB62′c 5 μl SB62c/5 μg QS21/5 μg 3D-MPL GR 5 2 μg Alum50 μg AlOH₃

Immunisation Scheme:

Animals were immunised intramuscularly in the leg (50 μl for all groupsexcept for group 5 where 100 μl was injected) at days 0, 21, 42 and 63.Blood was taken from the retroorbital sinus at various time points postimmunisations. On day 77, animals from each group were sacrificed,spleens and lymph nodes draining the site of injection (iliac lymphnodes) were taken out for in vitro restimulation. Pools of 3 or 4spleens and 1 pool of 10 LN were obtained for each group and treatedseparately in the in vitro assays.

Mouse Serology

Quantitation of anti-HBs antibody was performed by Elisa using HBsurface antigen as coating antigen. Antigen and antibody solutions wereused at 50 μl per well. Antigen was diluted at a final concentration of1 μg/ml in PBS and was adsorbed overnight at 4° C. to the wells of 96wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). Theplates were then incubated for 1 hr at 37° C. with PBS containing 1%bovine serum albumin and 0.1% TWEEN 20 (saturation buffer). Two-folddilutions of sera (starting at 1/100 dilution) in the saturation bufferwere added to the HBs-coated plates and incubated for 1 hr 30 min at 37°C. The plates were washed four times with PBS 0.1% TWEEN 20 andbiotin-conjugated anti-mouse IgG1, IgG2a, IgG2b or Ig (Amersham, UK)diluted 1/1000 in saturation buffer was added to each well and incubatedfor 1 hr 30 min at 37° C. After a washing step,streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted1/5000 in saturation buffer was added for an additional 30 min at 37° C.Plates were washed as above and incubated for 20 min with a solution ofo-phenylenediamine (Sigma) 0.04% H₂O₂ 0.03% in 0.1% TWEEN 20 0.05Mcitrate buffer pH4.5. The reaction was stopped with H₂SO₄ 2N and read at492/620 nm. ELISA titers were calculated from a reference by SoftmaxPro(using a four parameters equation) and expressed in EU/ml.

T Cell Proliferation

2 weeks after the second immunisation, mice were killed, spleen andlymph nodes were removed aseptically in pools (3 or 4 organs per poolfor splenic cells, 1 pool of 10 organs for LNC). Cell suspensions wereprepared in RPMI 1640 medium (GIBCO) containing 2 mM L-glutamine,antibiotics, 5×10⁻⁵ M 2-mercaptoethanol, and 1% syngeneic normal mouseserum. Cells were cultured at a final concentration of 2×10⁶ cells/ml(for LNC or SPC) in 200 μl in round-bottomed 96 well-plates withdifferent concentrations (10-0.03 μg/ml) of S,L* antigen (25D84). Eachtest was carried out in quadriplicate. After 96 hr of culture at 37° C.under 5% CO2, the cells were pulsed for 18 hr with 3H-Thymidine(Amersham, UK, 5 Ci/mmol) at 0.5 MC1/well and then harvested on fibreglass filters with a cell harvester. Incorporated radioactivity wasmeasured in a liquid scintillation counter. Results are expressed in cpm(mean cpm in quadriplicate wells) or as stimulation indices (mean cpm incultures of cells with antigen/mean cpm in cultures of cells withoutantigen).

Cytokine Production

2 weeks after the second immunisation, mice were killed, spleen andlymph nodes were removed aseptically in pools (3 or 4 organs per poolfor splenic cells, 1 pool of 10 organs for LNC). Cell suspensions wereprepared in RPMI 1640 medium (GIBCO) containing 2 mM L-glutamine,antibiotics, 5×10⁻⁵ M 2-mercaptoethanol, and 5% foetal calf serum. Cellswere cultured at a final concentration of 2.5 to 5×10⁶ cells/ml(respectively for LNC or SPC) in 1 ml, in flat-bottomed 24 well—withdifferent concentrations (1-0.01 μg/ml) of S,L* (25D84). Supernatantswere harvested 96 hrs later and frozen until tested for the presence ofIFNg and IL-5 by Elisa.

IFN-γ Production

Quantitation of IFNγ was performed by Elisa using reagents from Genzyme.Samples and antibody solutions were used at 50 μl per well. 96-wellmicrotiter plates (Maxisorb Immuno-plate, Nunc, Denmark) were coatedovernight at 4° C. with 50 μl of hamster anti-mouse IFNg diluted at 1.5μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37° C. with 100 μl of PBS containing 1% bovine serum albumin and 0.1%TWEEN 20 (saturation buffer). Two-fold dilutions of supernatant from invitro stimulation (starting at 1/2) in saturation buffer were added tothe anti-IFNg-coated plates and incubated for 1 hr 30 at 37° C. Theplates were washed 4 times with PBS TWEEN 0.1% (wash buffer) andbiotin-conjugated goat anti-mouse IFNg diluted in saturation buffer at afinal concentration of 0.5 μg/ml was added to each well and incubatedfor 1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham)diluted 1/10000 in saturation buffer was added for 30 min at 37° C.Plates were washed as above and incubated with 50 μl of TMB (Biorad) for10 min. The reaction was stopped with H₂SO₄ 0.4N and read at 450 nm.Concentrations were calculated using a standard curve (mouse IFNγstandard) by SoftmaxPro (four parameters equation) and expressed inpg/ml.

IL-5 Production

Quantitation of IL5 was performed by Elisa using reagents fromPharmingen. Samples and antibody solutions were used at 50 μl per well.96-well microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark) werecoated overnight at 4° C. with 50 μl of rat anti-mouse IL5 diluted at 1μg/ml in carbonate buffer pH 9.5. Plates were then incubated for 1 hr at37° C. with 100 μl PBS containing 1% bovine serum albumin and 0.1% TWEEN20 (saturation buffer). Two-fold dilutions of supernatant from in vitrostimulation (starting at 1/2) in saturation buffer were added to theanti-IL5-coated plates and incubated for 1 hr 30 at 37° C. The plateswere washed 4 times with PBS TWEEN 0.1% (wash buffer) andbiotin-conjugated rat anti-mouse IL5 diluted in saturation buffer at afinal concentration of 1 μg/ml was added to each well and incubated for1 hr at 37° C. After a washing step, AMDEX conjugate (Amersham) diluted1/10000 in saturation buffer was added for 30 min at 37° C. Plates werewashed as above and incubated with 50 μl of TMB (Biorad) for 15 min. Thereaction was stopped with H₂SO₄ 0.4N and read at 450 nm. Concentrationswere calculated using a standard curve (recombinant mouse IL5) bySoftmaxPro (four parameters equation) and expressed in pg/ml.

CTL Induction

2 weeks after the second immunisation, mice were killed, spleens wereremoved aseptically in pools of 3 or 4 mice (2 pools of 3 and one poolof 4 mice per group). Cell suspensions were prepared in RPMI 1640 medium(GIBCO) containing 2 mM L-glutamine, antibiotics, 5×10⁻⁵ M2-mercaptoethanol, and 5% foetal calf serum. Cells were cultured at afinal concentration of 2×10⁶ cells/ml in 10 ml medium containing 2 μg/mlSL* and 1.25% ConA sup (25 cm² Falcon flasks) and incubated for 8 daysat 37° C. under 5% CO2.

CTL Assay

The day before the CTL assay (d7), target cells were prepared byincubation of P815 cells (10⁶ cells/ml) with S,L* (batch 25D84) orpeptide S28-39 at 10 μg/ml. Following 1 hr incubation in 15 ml Falcontubes in a small volume, cells are transferred to 24 well plates andincubated ON at 37° C.

The day of the assay, 2×10⁶ S,L* and S₂₈₋₃₉ pulsed P815 cells and P815-Sare centrifugated, resuspended in 50 μl FCS and incubated with 75 μl⁵¹Cr (375 μCi) for 1 hr at 37° C. (shaking every 15′). Cells are thenwashed 4 times with 10 ml complete medium and incubated for 30′ at 4° C.following the 4th wash. Cells are then centrifugated and resuspended ata concentration of 2×10⁴ cells/ml.

Effector cells are then centrifugated, counted and resuspended at 2×10⁶cells/ml. Three fold serial dilutions of effector cells are done in 96V-bottomed plates, starting at a concentration of 2×10⁵ cells/well/100μl.

2×10³ target cells in 100 μl are added to effector cells in triplicate.Spontaneous and maximum release are assessed by incubating target cellsrespectively with medium or Triton X100 3%.

Plates are centrifugated 3′ at 700 rpm and incubated for 4 hrs at 37° C.Following the incubation time, 50 μl of supernatant is transferred fromeach well to Luma-plates and dryed overnight before counting inTop-count scintillation counter.

Results are expressed as specific lysis and calculated as follow:

% SR=(mean cpm sample−mean cpm medium/mean cpm max−mean cpm medium)×100

Results Serology

Humoral responses (Ig and IgG isotypes) were measured by ELISA using HBsurface antigen as coating antigen. Only data from the 21 days post IItime point are shown. These results are given in FIGS. 1 and 2.

FIG. 1, Shows Hbs specific Ig antibody responses measured on bothindividual mouse sera, and group average, 14 days post II.

FIG. 2, Shows the sub-isotype distribution of Hbs specific IgG in theserum the vaccinated mice.

-   -   As can be seen in FIG. 1, SB62 related formulations induce much        higher antibody titers than the S,L* Alum formulation.    -   Statistical analysis on individual sera (Anoval test Newman        Keuls) show no significant difference in antibody titers induced        by SB62c and SB62′c or equally between the antibody titers        induced by SB62 and SB62′c. The resultant anti-S,L* antibody        titres are, therefore, independent of the squalene:QS21 ratio.    -   The sub-isotypic distribution profile (as shown in FIG. 2) is        comparable for all SB62 related formulations (25-30% IgG2a)        whereas Alum induce only 4% IgG2a.

Cell-Mediated Immune Responses

Cell-mediated immune responses (lymphoproliferation, IFNγ/IL5 productionand CTL) were measured at 14 days post IV after in vitro restimulationof splenic and iliac lymph nodes cells with S,L* antigen.

Cytokine Production

Cytokine production (IFN-γ and IL-5) has been measured following 96 h ofin vitro restimulation of splenic cells and iliac lymph node cells withS,L*. These results are depicted in FIGS. 3 to 6.

FIG. 3, Shows the results of IFN-γ production by splenic cells (mean ofdata obtained with three pools/group).

FIG. 4, Shows the results of IL-5 production by splenic cells (mean ofdata obtained with three pools/group).

FIG. 5, Shows the results of IFN-γ production by Iliac lymph node cells(mean of data obtained with three pools/group).

FIG. 6, Shows the results of IL-5 production by Iliac lymph node cells(mean of data obtained with three pools/group).

TABLE 2 Ratio of IFN-γ:IL-5 producing cells detected in splenic cellsGroups Restimulation GR 1 GR 2 GR 3 GR 4 GR 5 S,L* 10 μg/ml 22.9 10.751.7 17.0 0.9

Discussion

-   -   Smaller amounts of emulsion are beneficial to IFN-γ production.        Very high levels of INF-γ are produced after by restimulation of        splenic cells from animals immunised with formulations        containing the low ratio emulsion. These levels are        significantly greater than those obtained after vaccination with        corresponding formulations using a full dose emulsion. The        strongest IFN-γ production is obtained after restimulation of        splenic cells from animals immunised with S,L* SB62 and SB62′c.    -   The beneficial effect of the low ratio formulations (groups 3        and 4 on FIGS. 5 and 6) are much more marked when looking at        cells derived from the draining lymph node (ileac lymph node)        compared to those taken from the spleen.    -   An IFN-γ:IL-5 ratio >1 clearly suggests that a pro TH1 response        is induced by all SB62 related formulations (see table 1).    -   Higher levels of IL-5 are produced by animals immunised with        S,L* SB62c formulations than S,L* SB62 formulations not        containing cholesterol. S,L* Alum immunised animals produce the        highest levels of IL-5.    -   A stronger IFN-γ production is observed when the low ratio        squalene:QS21 formulations (SB62′ and SB62′c) are used.

Cytotoxic T Cell Response

The anti-S,L* CTL responses are given in FIG. 7.

FIG. 7, Shows the CTL activity of splenic T-cells stimulated in vitrofor 7 days with S,L* antigen (mean % specific lysis of three pools).

Discussion of CTL Results

-   -   Specific lysis is obtained with all oil in water emulsion        formulations.    -   A stronger CTL response is observed with formulations containing        SB62′ emulsions when looking at limiting E/T ratio such as 3/1.

Conclusions

1. The strongest IFN-γ production is observed following immunisationwith SB62′ emulsions.2. A slightly better CTL response is induced by formulations containingSB62′ emulsions in comparison to the corresponding formulation using afull dose emulsion.3. The TH1 type profile of the immune response induced by all SB62related formulations is further confirmed by the IFN-γ/IL-5 ratio.4. No significant difference is observed between antibody titers inducedfollowing immunisation with SB62c full dose or SB62′c.5. No significant difference is observed between antibody titers inducedfollowing immunisation with SB62c and SB62′.6. A comparable isotypic profile (25-30% IgG2a) is obtained with allSB62 related formulations suggesting the induction of a TH1 type HBsspecific immune response.

TABLE 3 Summary table showing the results from example 2. ImmuneFormulations containing S, L* parameter SB62 SB 62c SB62′ SB62′c Alum Abtiters +++ +++ ++ +++ + TH type TH1 TH1 TH1 TH1 TH2 (% IgG2a) (29) (26)(29) (30) (4) IFN-γ (SPC) + ++ +++ ++++ + IL-5 (SPC) − + + ++ +++CTL + + ++ ++ −

EXAMPLE 3 Immunogenicity Studies with Malaria Antigens TRAP and RTS,S

Immunisation experiments using the Plasmodium falciparum Malariaantigens TRAP and RTS,S in combination with various adjuvants, eachbased on an oil in water emulsion system. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(S) antigen of hepatitis B virus. It's full structure is disclosed inthe International Patent Application No. PCT/EP92/02591, published underNumber WO 93/10152 claiming priority from UK patent application No.9124390.7. When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S.

TRAP antigens are described in the International Patent Application No.PCT/GB89/00895, published under WO 90/01496. TRAP antigens arepolypeptides, so called Thrombospondin Related Anonymous Proteins, whichshare homology with various P. falciparum proteins.

Different adjuvant formulations, using the emulsion systems as describedin example 1, with differing ratios of squalene:QS21, and optionallycomprising cholesterol (QS21:cholesterol ratio w/w of 1:10), werecombined with the malaria antigens and compared in their ability toinduce humoral and cell mediated immune responses (T-cell proliferationand cytokine production). SB62 was formulated together with the antigenat a high (240:1, SB62) or low (48:1, SB62′) ratio of squalene:QS21,optionally with the addition of cholesterol (c).

Groups of 5 mice (six week old female mice, strain C57/BL6×CBA/J [H−2k]) were immunised twice (in 2×50 μl volumes) in the hind foot-pad, 14days apart, with either 10 μg RTS,S or 4 μg TRAP combined with variousoil in water emulsion systems (SB62). 14 days following the secondimmunisation the production of cytokines (IL5 and IFN-γ) and T-cellproliferation was analysed after in vitro restimulation of spleen andlymph nodes cells with the malaria antigens. Antibody response to RTS,Sand TRAP and the isotypic profile that was induced was investigated byELISA.

TABLE 4 Groups of animals and vaccine formulations used in example 3.Group No. Antigen Adjuvant 1 RTS,S SB62/QS21/3D-MPL 2 TRAPSB62/QS21/3D-MPL 3 RTS,S/TRAP SB62/QS21/3D-MPL 4 RTS,S AlOH/QS21/3D-MPL5 RTS,S/TRAP AlOH/QS21/3D-MPL 6 RTS,S SB62c/QS21/3D-MPL 7 RTS,S/TRAPSB62c/QS21/3D-MPL 8 RTS,S SB62′/QS21/3D-MPL 9 RTS,S/TRAPSB62′/QS21/3D-MPL 10 — SB62/QS21/3D-MPL 11 Vac. Vir. 3D7 Footnotes:SB62 - oil in water emulsion full dose SB62′-oil in water emulsionexemplified in the figures as SB62 ⅕th dose SB62c or SB62′c - oil inwater emulsion (either dose) plus cholesterol in the oil phase. Vac.Vir. 3D7 = a recombinant vaccinia virus construct expressing CS proteinand administered at 10⁶ PFU per mouse.

T-Cell Proliferation

Spleen or popliteal lymph node cells were aseptically removed andwashed. 100 μl of cells in RPMI medium (1% heat-inactivated normal mouseserum, NMS) containing 2×10⁶/ml of cells were cultured in round bottomedplates in the presence of RTS,S or TRAP antigens. Following stimulationfor 96 hours with 0.1, 0.5, and 2.5 μg of antigen, or 48 hours with 2μg/ml ConA, the cells were labelled with ³H-Thymidine (1 μCi/well) for16 hours before harvesting and counting in a β-counter.

RPMI Medium:

RPMI 1640 without L-glutamine (Life technologies No. 31870025), 2 mML-glutamine (Life technologies No. 25030024), 50 μM 2-Mercaptoethanol(Life technologies No. 11360039), 1 mM Sodium Pyruvate (Lifetechnologies No. 11360039), 1×MEM non essential amino acids (10× stock,Life technologies No. 11140035), 100 IU/ml penicillin-100 μg/mlstreptomycin (Life technologies No. 15140114).

Cytokine Detection

Spleen or popliteal lymph node cells were aseptically removed andwashed. 1000 μl of cells in RPMI medium (5% heat-inactivated fetal calfserum, FCS) containing 5×10⁶/ml of cells were cultured in 24 well flatbottomed plates in the presence of RTS,S or TRAP antigens. The plateswere then incubated (37° C., 5% CO₂) for a number of hours with 0.5, and2.5 μg of antigen, or 4 μg/ml final of ConA.

The length of time that the cells were incubated depended on theparticular cytokine to be detected, IL-2 was stimulated for 72 hours,IL-5 was 72 or 96 hours, and IFN-γ was 96 hours. Each cytokine wasdetected using commercially available ELISA kits (IL-2 and IFN-γ, DuosetGenzyme No. 80-3573-00 and 80-3931-00 respectively; IL-5 was detectedusing the Pharmingen kit).

Serology

Antibodies directed against TRAP were analysed using a sandwich ELISA. Asheep anti-TRAP antiserum was coated onto ELISA plates which was used tocapture TRAP antigen added at 0.5 μg/ml. Titrations of pooled serum fromthe experimental groups were added and incubated. Finally, biotinylatedanti-mouse isotype-specific antibodies followed bystreptavidin-peroxidase, were used to detect bound TRAP-specificantibodies.

Anti HBV humoral responses were analysed by a direct ELISA, HBsAg wascoated onto the ELISA plate at 1 μg/ml. Pooled serum from the differentexperimental groups were titrated and bound antibodies were detected asdescribed above.

Results Proliferation of Lymphoid Cells in Response to Antigen

The proliferative responses in response to antigen can be seen in FIGS.8 to 11. All vaccine preparations stimulated cells in the localpopliteal lymph node which were capable of proliferating in vitro inresponse to antigen, the magnitude of which was independent of theaddition of cholesterol.

All vaccine preparations were capable of stimulating splenic cells whichproliferated in vitro in response to antigen. When considering thestimulation indices, the preparations which elicited the highestresponses in the spleen were the ones having the low ratio squalene:QS21(48:1 or ⅕th dose SB62).

FIG. 8, Shows the proliferative responses of popliteal lymph node cells(in raw counts per minute (CPM) form) derived from the experimentalgroups after stimulation with TRAP and RTS,S antigens.

FIG. 9, Shows the proliferative responses of splenic cells (in rawcounts per minute (CPM) form) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 10, showing the proliferative responses of popliteal lymph nodecells (Stimulation index) derived from the experimental groups afterstimulation with TRAP and RTS,S antigens.

FIG. 11, Shows the proliferative responses of splenic cells (Stimulationindex) derived from the experimental groups after stimulation with TRAPand RTS,S antigens.

Discussion of Proliferation Results

FIGS. 1 and 2, clearly show that all of the vaccine formulationsstimulate lymphoid cells which are capable of proliferating in vitro inthe presence of antigen in a dose dependent manner. The raw cpm datasuggests that the inclusion of cholesterol in the adjuvant formulationshas no effect on the magnitude of the proliferative responses (forexample a comparison between groups 1 and 6, termed RTS,S/MPL/QS21/SB62and RTS,S/MPL/QS21/SB62c respectively).

Examination of the cpm together with the stimulation index results(obtained by dividing the raw cpm for antigen specific proliferation bythat derived from non-antigen specific proliferation (medium alone))shows that the vaccine formulation which generates the highestproliferative responses depends on the origin of the lymphocytemeasured. The adjuvant formulations containing the low ratio ofsqualene:QS21 (48:1) generate the highest proliferative responses in thespleen.

In Vitro Cytokine Production Upon Stimulation with Antigen

Cytokine production, measured in vitro in response to antigen, can beboth a quantitative and qualitative measure of the induction of immuneresponses in vivo. In general high levels of IFN-γ and IL-2 are taken tobe a measure of Th1-type immune responses and IL-5 is considered to be aTh2-type cytokine. The following figures (FIGS. 12 to 14) demonstratethe use of SB62′, containing a reduced ratio of squalene:QS21 (termedSB62 ⅕th dose), had a marked effect in enhancing the production of IFN-γ(FIG. 6).

Further, there is evidence that the addition of cholesterol has noqualitative or quantitative effects on the cytokine profile produced invitro in response to antigen. This effect may have significantconsequences in the induction of Th1-type immune responses and alsoimmunotherapeutics.

FIG. 12, shows the IL-2 production of spleen cells after stimulationwith TRAP or RTS,S antigen 14 days after VII.

FIG. 13, shows the IFN-γ production by spleen cells after stimulationwith TRAP or RTS,S antigen 14 days after VII.

FIG. 14, shows the IL-5 production by spleen cells after stimulationwith TRAP or RTS,S antigen 14 days after VII.

Serology

Another measure of immunity that can correlate to a Th1-type, oralternatively a Th2-type, immune response is the IgG sub-isotype whichis elicited. A preferential stimulation of the IgG1 sub-isotype isgenerally taken to be a measure of the induction of a Th2-type immuneresponse, and conversely IgG2a and IgG2b is taken to be a measure of aTh1 type immune response.

ELISA studies were performed on pooled mouse serum and the mid-pointtitres for both the HBsAg and TRAP specific antibodies were ascertained.From these figures, the ratio of the antigen specific IgG1 and IgG2amid-point titres was calculated and taken to be a measure of the Th1/Th2balance of the humoral immune response (the results are shown in table4).

TABLE 4 The ratio of IgG1:IgG2a, representing the Th1/Th2 balance. Aratio <1 represents a Th1-type immune response, a ratio of >1 indicatinga Th2-type response. Ratio of mid-point titres IgG1:IgG2a Group HBsAgTRAP 1 0.44 2 0.36 3 1.46 1.68 4 0.37 5 0.39 11.83 6 0.28 7 0.2 7.21 80.66 9 0.3 0.77

Discussion of Serological Results

Pools of mouse serum were analysed from each group and were found tohave successfully stimulated HBsAg and TRAP specific antibodies. Ingeneral, antibody mid-point titres against HBsAg were higher than thosefound against TRAP. The isotype distribution differed between the twoantigens. RTS,S in all formulations elicited a clear Th1 pattern, asindicated by an IgG1:IgG2a ratio below 1.

In contrast, TRAP-specific antibodies exhibited a Th2-type isotypepattern. The only exceptions to this observation were groups 2, whoreceived TRAP alone, and group 9, who received TRAP/RTS,S in a SB62′formulation (containing a low ratio of squalene:QS21, termed SB62 ⅕thdose). The use of SB62′ may, therefore, be useful in the design ofTh1-inducing vaccines with antigens which are known to preferentiallyinduce Th2-type immune responses.

EXAMPLE 4 Immunological Studies Using a Murine Tumour Regression Model

This experiment investigated the potential use of oil in water emulsionadjuvants for the therapeutic treatment of Human Papilloma virus (HPV)expressing tumors. Tumor cells (TC1), known to express the E7 protein ofHPV 16, were innoculated into C57BL/6 6-8 weeks old mice. These tumorcells if left untreated grew into tumors of measurable size. Thepotential of E7 comprising vaccines, based on oil in water emulsionadjuvants, to prevent the establishment of these tumors wasinvestigated. The therapeutic potential of various oil in wateremulsions (for details see example 1) SB62 full-dose, SB62 ⅕, SB62cfull-dose, and SB62c ⅕ in combination with ProtD1/3 E7 HPV16 recombinantantigen, was evaluated in the TC1-E7 tumor model. Further, thecontribution of vaccination schedules were compared.

Briefly, groups of 8-10 C57BL/6 mice were challenged with 5×10⁵ TC1tumour cells (in the flank). The groups of mice were then immunizedintra-footpad with 5 μg ProtD1/3 E7 combined with various formulations,7 and 14 days after a subcutaneous tumor challenge.

Other vaccination shemes were compared: 2 vaccinations with 5 μgProtD1/3 E7 in SB62 (days 14 and 21 after the tumor challenge); and 4vaccinations with 5 μg ProtD1/3 E7 in SB62 (7, 14, 21, 28 days aftertumor challenge).

Antibody responses to E7 were monitored by ELISA at time points, 2 and 4weeks post second vaccination. Lympho-proliferative response wasanalyzed by in vitro restimulation of spleen and lymph nodes cells for72 hrs with the protein E7 (10 μl, 0.1 μg/ml) 2 and 4 weeks post secondvaccination. CTL responses were measured after in vitro re-stimulationof spleen cells with irradiated tumor cells (TC1) or an E7-derivedpeptide. The Chromium release assay was performed on TC1 cells, on asyngeneic tumor cell line: EL4 pulsed or not with an E7-derived peptideor infected either with a E7 recombinant vaccinia virus or with the wildtype vaccinia virus.

TABLE 5 Groups of mice Vaccination schedule Group (days after challenge)Antigen (HPV 16) Exipient a 7, 14 — PBS b 7, 14 ProtD1/3 E7 PBS c 7, 14ProtD1/3 E7 DQ d 7, 14 — DQ e 7, 14 ProtD1/3 E7 SB62 f 7, 14 — SB62 g 7,14 ProtD1/3 E7 SB62′ h 7, 14 — SB62′ i 7, 14 ProtD1/3 E7 SB62c j 7, 14 —SB62c k 7, 14 ProtD1/3 E7 SB62′c l 7, 14 — SB62′c m 7, 14, 21, 28ProtD1/3 E7 SB62 n 14, 21 ProtD1/3 E7 SB62

Therapeutic Experiments: Protocol

-   -   5×10⁵ TC1-E7 expressing tumor cells were injected subcutaneously        (200 μl) in the flank of C57BL/6 immunocompetent mice    -   Vaccinations were performed at either 7, 14, 21, or 28 days        after the tumor challenge, with 5 μg ProtD 1/3 E7 HPV16 injected        intra-footpad (100 μl:50 μl/footpad). Each vaccine was        formulated in the presence of different adjuvants: SB62, SB62c,        SB62′ or SB62′c.    -   2 and 4 weeks after the second immunization, mice were killed        and spleens or popliteal lymph nodes were taken and assayed in        lymphoproliferation or CTL assays.

Comparative Liposome-Based Formulations (DQ)

ProtD1/3-E7 antigen (5 μg) was incubated 30 min with MPL (5 μg) beforebuffer addition as a mix of 10 fold concentrated PBS pH 7.4 and H₂O.After 30 min, QS21 (5 μg) was added to the formulation mixed withliposomes in a weight ratio QS21/Cholesterol of 1:5 (referred to as DQ).50 μg/ml of thiomersal were added to the formulation as preservative 30min after addition of the QS21. All incubations were carried out at roomtemperature with agitation.

Cell Lines

TC1 (obtained from the John Hopkin's University), or EL4 cells weregrown in RPMI 1640 (Bio Whittaker) containing 10% FCS and additives: 2mM L-Glutamine, 1% antibiotics (10000 U/ml penicillin, 10000 μg/mlstreptomycin) 1% non essential amino acid 100×, 1% sodium pyruvate(Gibco), 5 10e-5 M 2-mercaptoethanol. Before injection into the flank ofthe mice, the TC1 cells were trypsynized and washed in serum freemedium.

Tumor Growth

Individual tumor growth was followed over time. The 2 main diameters (A,B) were measured using calipers twice a week, A×B represents the “tumorsurface” and is expressed as the average of the 5 values in each group.

In Vitro Lymphoproliferation

Lymphoproliferation was performed on individual spleens and on lymphnode pools. The cell suspension were incubated with Tris-bufferedammonium chloride for 4 min at 4° C. in order to lyse the red bloodcells. 2×10⁵ spleen cells or popliteal lymph node cells were plated intriplicate, in 96 well microplate, in RPMI medium containing 1% normalmouse serum. After 72 hrs incubation with different amounts of E7(10-1-0.1 μg/ml), 100 μl of culture supernatant were removed andreplaced by fresh medium containing 1 μCi ³H-thymidine. After pulsingfor 16 hrs, the cells were harvested onto filter plates. Incorporatedradioactivity was counted in a β counter. Results are expressed incounts per minute (CPM, mean of triplicate wells) or as stimulationindexes (mean CPM in cultures with antigen÷mean CPM in cultures withoutantigen).

CTL assay

2×10⁶ spleen cells were co-cultured with 2×10⁶ irradiated (18000 rads)TC1 cells for 7 days. Target cells were either Cr⁵¹ (DuPont NEN 37MBq/ml) loaded (1 hr at 37° C.) TC1 cells or EL4 cells (syngeneic tumorcells) infected with an E7 recombinant vaccinia virus (received from T.C. Wu from the John Hopkins University). The results derived from thesecells were compared to those from EL4 targets which had been infectedwith the wild type vaccinia virus (Vaccinia infection is performed at aMOI of 10 in a small volume of serum free medium, for 1H, at 37° C. in aC02 incubator. Fresh medium was added and cells were incubated overnightbefore use). 10 μg/ml of E7-derived peptide (49-57) (QCB) was used topulse EL4 cells for 1 hr at 37° C. during the Cr⁵¹ loading of the cells.2000 target cells were added to each well of 96 well plate (V bottomnunc 2-45128) with 100/1 being the highest Effector/target ratio.Controls for spontaneous or maximal Cr⁵¹ release were performed insextuplet and were targets in medium or in triton 1.5%. All plates weregently centrifuged and incubated for 4 hrs at 37 in 7% CO2. 50 μl of thesupernatant was deposed on 96w Lumaplate (Packard) let dry O/N andcounted in a Top Count counter. Data is expressed as percent specificlysis which is calculated from the c.p.m. by the formula (experimentalrelease−spontaneous release)÷(maximal release−spontaneous release)×100.

Serology

Quantitation of anti E7 antibody was performed by Elisa using E7 ascoating antigen. Antigen and antibody solutions were used at 50 μl perwell. Antigen was diluted at a final concentration of 3 μg/ml incarbonate buffer ph9.5 and was adsorbed overnight at 4° c. to the wellsof 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark).The plates were then incubated for 1 hr at 37° c. with PBS containing 1%bovine serum albumin and 0.1% Tween 20 (saturation buffer). Two-folddilutions of sera (starting at 1/100 dilution) in the saturation bufferwere added to the E7-coated plates and incubated for 1 hr 30 min at 37°c. The plates were washed 3 times with PBS 0.1% Tween 20 andbiotin-conjugated anti-mouse IgG1, IgG2a or IgG2b or IgGtot (Amersham,UK) diluted 1/5000 in saturation buffer was added to each well andincubated for 1 hr 30 min at 37° c. After a washing step,streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted1/5000 in saturation buffer was added for an additional 30 min at 37° c.Plates were washed as above and incubated for 10 min with TMB(tetra-methyl-benzidine) The reaction was stopped with H₂SO₄ 4N and readat 450 μm. Midpoint dilution were calculated by SoftmaxPro (using a fourparameters equation).

Immunohistochemistry

Tumours were excised and fixed in acetone and paraformaldehyde prior tosectioning. The 5 μm thick cryostat secretions were then investigatedand stained for CD4, CD8, and CD3 expressing T-cells infiltration. Priorto the addition of the staining monoclonal antibodies, the sections werewashed and saturated with 0.5% bovine serum albumin (BSA), 5% normalrabbit serum (NRS) in PBS. After this step the rat anti-CD3, CD4, andCD8 monoclonal antibodies were added and incubated overnight at 4 C. Thesections were then washed and the binding of the rat monoclonalantibodies was revealed with biotinylated rabbit anti-rat Ig. Afterincubation for 30 mins, at room temperature (RT), streptavidin-Horseradish peroxidase was added and incubated for another 30 mins at RT. Thebinding of the streptavidin-Horse radish peroxidase was revealed withDAB for 10 minutes at RT. The sections were then counterstained withHematoxylin, and dehydrated with ethanol, isopropanol, and finallyxylol.

Results

Tumor growth (for a representation of the mean tumor growth/group seeFIG. 15)

FIG. 15, Shows mean tumour growth after challenge and vaccination ondays 7 and 14 with various ProtD1/3 E7 containing formulations.

FIG. 16, Shows the mean tumor growth observed over a period of 4 weeksfor the groups receiving the antigen in DQ and SB62 formulations, alsorepresented are the results comparing the different vaccinationschedules. These vaccines were administered on days 7 and 14; or days 14and 21; or days 7, 14, 21, and 28.

FIG. 16, Comparison with comparative formulations and other vaccinationschedules with the ProtD1/3 E7 antigen.

Discussion of the Tumour Regression Studies

-   -   Vaccination with either ProtD1/3E7 or adjuvant alone has no        effect on the growth of the TC1-E7 expressing tumour.    -   The analysis of individual tumor growth showed complete tumour        rejection in several groups:

Group Percentage tumour rejection c 40% e 40% g 60% j 20% l 10%

-   -    The best formulation to induce tumor rejection were formulated        with the low dose SB62′ oil in water emulsion.    -   A better therapeutic effect was observed after 4 vaccinations        than seen after 2. Analysis of individual tumor growth showed        that 60% of the animals completely rejected their tumor after 4        vaccinations with a SB62 based formulation whilst only 40% of        mice having received 2 vaccinations showed complete regression.    -   If the first vaccination was delayed until day 14 following the        tumor challenge, no complete rejection could be observed.        However, the tumor growth seemed to be abrogated.

Proliferation Results

-   -   No proliferative response was observed in this experiment either        with spleen or lymph node cells from mice that received ProtD1/3        E7 or adjuvants alone.    -   Antigen specific lymphoproliferation was increased in the groups        of mice that received protD 1/3 E7 in the presence of adjuvants.        High proliferative responses were observed with both DQ, and        SB62′ in the spleen. See FIGS. 17 and 18.

FIG. 17, Lymphoproliferation observed in spleen cells, 2 weeks after thesecond vaccination.

FIG. 18, Lymphoproliferation observed in spleen cells, 2 weeks after thesecond vaccination.

Serology

Anti E7 antibody response: IgG total and sub-isotypes (IgG1, IgG2a,IgG2b) were measured by ELISA using the E7 protein as coating antigen.The anti-E7 Ig titres observed 2 weeks after the second vaccination aregiven in table 6. FIG. 19, shows the relative percentage of thedifferent IgG isotypes in the serum of vaccinated mice 2 weeks after thesecond vaccination.

-   -   The weak antibody response induced after 2 vaccinations with the        ProtD1/3 E7 alone was strongly increased in animals that        received an adjuvant. The strongest antibody response was        obtained with SB62.    -   The predominant E7 specific antibody sub-isotype induced by all        of the vaccine formulations tested was IgG2b (80-90% of the        total IgGs).

TABLE 6 anti-E7 Ig titres observed 2 weeks after the second vaccinationIg sub-isotype titre Group IgG1 IgG2a IgG2b a 0 0 0 b 1420 0 4070 c 78501110 70170 d 0 0 0 e 11880 470 86610 f 0 0 0 g 13670 1580 62560 h 0 0 0i 13073 1650 89930 j 0 0 0 k 260 0 2630 l 0 0 0

FIG. 19, Relative percentage of the different IgG isotypes in the serumof vaccinated mice 2 weeks after the second vaccination.

CTL Results

-   -   A CTL response could be detected at time points 2 and 4 weeks        after the final vaccination.    -   No lysis was observed when mice received the protein or the        adjuvant alone. The best specific lysis was observed when mice        received the antigen in DQ or SB62′ (see table 7).

TABLE 7 Summary of CTL responses after stimulation of spleniclymphocytes with TC1 EL4 + EL7. Anti-E7 CTL Group (E:T ratio 100:1) a −b − c +++ d − e ++ f − g ++++ h − i + k ++ l +

Immunohistochemistry Results

Tumors were removed from the mice (2 mice per group) and sections werefrozen. Cryo section of tumors were stained with anti-CD4 and anti-CD8antibodies. The results for the observed tumor infiltration by CD4 andCD8+ve cells are given in Table 8.

TABLE 8 results of lymphocytic tumour infiltration after vaccinationwith Group Negative CD4+ve lymphocyte CD8+ve lymphocyte (Mouse No.)control infiltration infiltration a(1) − − +/− a(2) − − +/− b(1) − +/− +b(2) − − + c(1) − + +++ c(2) − + +++ d(1) − − +/− d(2) − +/− + e(1) −+/− +++ e(2) − +/− +++ f(1) − − +/− f(2) − − +/−

Conclusions

-   -   The regressing tumors, in the groups of mice that received the        ProtD-1/3 E7 in DQ or SB62, showed a massive infiltration of        with CD8+ cells and few CD4+ cells.    -   Tumors removed from the animals that received the PBS, antigen,        or adjuvants alone, did not contain any CD8+ve lymphocytic        infiltration.    -   Two vaccinations (on days 7, 14) with 5 μg ProtD 1/3 in        different SB62 based formulations induced the rejection of        pre-established E7 expressing tumors implanted at a distant        site.    -   Tumor rejection is associated with an anti E7 specific CTL        response. There is a trend to have a slightly better CTL        response in the individuals that rejected their tumors.    -   Immunochemistry showed a massive infiltration of CD8+ T cells in        tumors that regressed upon vaccination with ProtD1/3 E7+DQ and        SB62.    -   Two vaccinations (on days 114 and 21 post tumour challenge) with        5 μg ProtD 1/3 E7 HPV16 in SB62 reduced the growth of these        bigger tumors but do not induce complete regression.    -   Four vaccinations (days 7, 14, 21, and 28 post tumour challenge)        with 5 μg ProtD 1/3 E7 HPV16 in SB62 induced the complete        rejection of the established tumors in 60% of the animals. 40%        total rejection was observed after 2 vaccinations with the same        adjuvant.    -   The use of the low dose SB62′ adjuvant had no effect on the        magnitude of the anti-E7 antibody titres, yet induced the        highest level of splenic lymphocyte proliferation and anti-E7        CTL responses.

Overall Conclusions to the Invention:

It is clear from the examples above that the present inventionencompasses an oil in water emulsion which preferentially induces astrong Th1-type immune responses, especially IFN-γ production. Theseformulations have been demonstrated to stimulate immune responses to awide variety of antigens and therefore, it is envisaged that thispresent invention shall find utility in a wide variety of pathogens notlimited to those found herein.

EXAMPLE 5 Stabilisation of QS21 by Addition of Cholesterol

It has previously been described that QS21-His hydrolysis product ofQS21, that is no longer active as adjuvant. It is formed by cleavage ofthe QS21 molecule by OH⁻ from the aqueous solution. This reaction occurswhere the pH of the aqueous medium is above a value of 6.5, and isaccelerated by higher temperature. The oil-in-water emulsions describedin this patent application (for example SB62) are known to exhibit astabilising effect such that the hydrolysis of QS21 into QS21-Hisinhibited. Upon dilution of the oil in water emulsion in the presence ofconstant QS21, they lose this stabilising property and the QS21degenerates into the inactive QS21-H form. Surprisingly, emulsionscontaining additional Cholesterol, who at 1/1 ratio do not show animproved QS21 stability, maintain the stabilising effect even at a 1/5dilution.

QS21 and QS21-H are assayed directly into the emulsion. This is achievedby chemically derivatising the complete formulation, and by performing aselective extraction step that dissolves the QS21, but leaves mostinterfering matrix compounds behind. The assay is HPLC based, and thecompounds are dansylated. The dansylation is performed by drying down asample of the emulsion, and adding 100 μl of 3.5 mg Dansyl hydrazine/mlC/M 2/1 and 100 μl of 1:4 Acetic acid: C/M 2/1 in that order. Themixture is well vortexed and incubated at 60° C. for 2 hours. Thereaction mixture is dried in the Speedvac. It is reconstituted in 500 μl30% ACN in H2O, and centrifugated twice at 14000 rpm for two minutes.The supernatants are then collected in an autosampler tube. A standardcurve is obtained by preparing QS21 and QS21-H in a mixture thatcontains the same compounds as the emulsion under study.

The HPLC assay is ran on a Vydac 218TP54 5μ particle size C18 RP column,250*4.6 mm. Solvents are A:H2O+0.05% TFA (trifluoracetic acid) andB:Acetonitrile+0.05% TFA. The gradient table is:

Time (min) % A % B 0 70 30 2 70 30 15 50 50 17 50 50 17.1 10 90 19 10 9021 70 30 25 70 30

The Flow rate is 1 ml/min. Detection is in fluorescence, with excitationat 345 nm and emission at 515 nm. 50 μl is injected of both the sampleand the standards. The column heater is set to 37° C. for thisseparation. Peaks for QS21, QS21-iso and QS21-H are distinguished on thechromatogram.

A series of samples with the following composition were analysed:

Composition SB62 SB62c MPL QS21 SB62 250 μl — 50 μg 50 μg SB62′  50 μl —50 μg 50 μg SB62c — 250 μl 50 μg 50 μg SB62′c —  50 μl 50 μg 50 μg

Assay of QS21/QS21-H was performed after incubation of the samples atvarious time intervals and temperatures (4° C. and 37° C.). The data for1 month at 37° C. in this model correlate well with stability of QS21after prolonged storage at 4° C. (eg 2 years).

TABLE 9 HPLC QS21 assay: % of QS21-H generated over time 3 months (4°C.) + 3 months 6 months 7 days 1 month Composition (4° C.) (4° C.) (37°C.) (37° C.) SB62 1% 2% 3% 15% SB62′ 1% 1% 9% 31% SB62c 2% 2% 3% 17%SB62′c 2% 2% 3% 21%

This results shown in the table above shows clearly (both for 7 days and1 m) the effect of adding a sterol, in this case cholesterol, to SB62′in maintaining the stability of QS21.

1-29. (canceled)
 30. A composition comprising an oil in water emulsionand a saponin, wherein said oil is a metabolisable oil, characterised inthat the ratio of the metabolisable oil:saponin (w/w) is in the range of1:1 to 200:1.
 31. A composition as claimed in claim 30, characterisedthat the ratio of the metabolisable oil:saponin (w/w) is in the range of1:1 to 100:1.
 32. A composition as claimed in claim 30, characterised inthat the ratio of the metabolisable oil:saponin (w/w) is 48:1.
 33. Acomposition as claimed in claim 30, where the saponin is derived fromQuilA.
 34. A composition as claimed in claim 30, where the metabolisableoil is squalene.
 35. A composition as claimed in claim 30, furthercomprising a sterol.
 36. A composition as claimed in claim 35, where thesterol is cholesterol.
 37. A composition as claimed in claim 30, furthercomprising one or more additional immunomodulators.
 38. A composition asclaimed in claim 35, further comprising one or more immunomodulators.39. A composition as claimed in claim 37, wherein said immunomodulatoris selected from the group comprising: 3D-MPL and α-tocopherol.
 40. Acomposition as claimed in claim 38, wherein said immunomodulator isselected from the group comprising: 3D-MPL and α-tocopherol.
 41. Acomposition as claimed in claim 33, and further comprising 3D-MPL,characterised in that the ratio of QS21:3 D-MPL (w/w) is from 1:10 to10:1.
 42. A composition as claimed in claim 41, wherein the ratio ofQS21:3 D-MPL (w/w) is from 1:1 to 1:2.5.
 43. A composition as claimed inclaim 30, and further comprising cholesterol, and wherein the saponin isQS21, characterised in that the ratio of QS21:cholesterol (w/w) is inthe range of 1:1 to 1:20.
 44. A vaccine composition comprising acomposition as claimed in claim 30, further comprising an antigen orantigenic preparation.
 45. A vaccine composition as claimed in claim 44,where the antigen or antigenic preparation is prepared from the groupof: Human Immunodeficiency Virus; Herpes Simplex Virus type 1; HerpesSimplex Virus type 2; Human Cytomegalovirus; Hepatitis A, B, C or E;Respiratory Syncitial Virus, Human Papilloma Virus; Influenza Virus;Salmonella; Neisseria; Borrelia; Chlamydia; Bordetella; TB; EBV;Plasmodium and Toxoplasma.
 46. A vaccine composition as claimed in claim44, wherein the antigen or antigenic preparation is a combination of theMalaria antigens RTS,S and TRAP.
 47. A vaccine composition as claimed inclaim 44, where the antigen or antigenic preparation is a tumour or hostantigen, or an immunogenic protein or peptide thereof.
 48. A compositionas claimed in claim 30, wherein the oil in water emulsion comprises oildroplets which have a diameter which is less than 1 micron.
 49. Acomposition as claimed in claim 30, where in the oil in water emulsioncomprises oil droplets which are in the range of 120 to 750 nm indiameter.
 50. A composition as claimed in claim 30, wherein the oil inwater emulsion comprises oil droplets which are in the range of 120 to600 nm in diameter.
 51. A vaccine composition as claimed in any one ofclaims 44 to 47, that invokes a cytolytic T-cell response in a mammal tothe antigen or antigenic composition.
 52. A vaccine composition asclaimed in claim 44 that stimulates interferon-γ production in mammal tothe antigen or antigenic composition.
 53. A method for manufacturing avaccine as claimed in claim 44, comprising admixing an oil in wateremulsion; QS21; cholesterol; 3D-MPL; α-tocopherol; and an antigen orantigenic preparation.
 54. A method of treating an individualsusceptible to or suffering from a disease comprising the step ofadministering a vaccine composition as claimed in claim
 44. 55. A methodof stabilising a saponin present in a composition of claim 30,comprising the addition of a sterol into the oil phase of said oil inwater emulsion.
 56. A method as claimed in claim 55, wherein the saponinis QS21.
 57. A method as claimed in claims 55 or 56 wherein the sterolis cholesterol.
 58. A composition as claimed in claim 33, where thesaponin is QS21.
 59. A composition as claimed in claim 30, wherein saidmetabolisable oil is α-tocopherol.
 60. A composition as claimed in claim30, wherein said metabolisable oil is α-tocopherol and squalene.
 61. Acomposition as claimed in claim 59, further comprising the saponin QS21.62. A composition as claimed in claim 61, wherein the ratio ofoil:sponin (w/w) is in the range of 100:1.